CN117143963A - Method for recovering nucleic acid in PCR product by ethanol-sodium acetate method - Google Patents
Method for recovering nucleic acid in PCR product by ethanol-sodium acetate method Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 57
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 20
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 20
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 20
- VIGBIBDAVDHOTP-UHFFFAOYSA-M sodium;ethanol;acetate Chemical compound [Na+].CCO.CC([O-])=O VIGBIBDAVDHOTP-UHFFFAOYSA-M 0.000 title abstract description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 63
- 229960000789 guanidine hydrochloride Drugs 0.000 claims abstract description 35
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 claims abstract description 35
- 239000004353 Polyethylene glycol 8000 Substances 0.000 claims abstract description 21
- 235000019446 polyethylene glycol 8000 Nutrition 0.000 claims abstract description 21
- 229940085678 polyethylene glycol 8000 Drugs 0.000 claims abstract description 21
- 239000001632 sodium acetate Substances 0.000 claims abstract description 16
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims abstract description 15
- 235000017281 sodium acetate Nutrition 0.000 claims abstract description 15
- 239000000701 coagulant Substances 0.000 claims abstract description 14
- 238000003916 acid precipitation Methods 0.000 claims abstract description 3
- 238000000926 separation method Methods 0.000 claims abstract description 3
- 239000000047 product Substances 0.000 claims description 67
- 238000005119 centrifugation Methods 0.000 claims description 38
- 239000007788 liquid Substances 0.000 claims description 28
- 235000019441 ethanol Nutrition 0.000 claims description 19
- 239000006228 supernatant Substances 0.000 claims description 16
- 239000000203 mixture Substances 0.000 claims description 10
- 238000001035 drying Methods 0.000 claims description 2
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims 1
- 238000011084 recovery Methods 0.000 abstract description 46
- 230000001737 promoting effect Effects 0.000 abstract description 2
- 238000002474 experimental method Methods 0.000 description 25
- 239000000523 sample Substances 0.000 description 19
- 239000002244 precipitate Substances 0.000 description 13
- 230000007812 deficiency Effects 0.000 description 12
- 238000001556 precipitation Methods 0.000 description 10
- 239000000243 solution Substances 0.000 description 9
- 239000007984 Tris EDTA buffer Substances 0.000 description 6
- 238000000246 agarose gel electrophoresis Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 238000011027 product recovery Methods 0.000 description 5
- 238000004062 sedimentation Methods 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000008188 pellet Substances 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 238000001976 enzyme digestion Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 229920000936 Agarose Polymers 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 239000006185 dispersion Substances 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 230000002195 synergetic effect Effects 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 239000012521 purified sample Substances 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 229910001415 sodium ion Inorganic materials 0.000 description 1
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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Abstract
The application provides a method for recovering nucleic acid in PCR products by ethanol-sodium acetate method, which comprises adding guanidine hydrochloride with final concentration of 1-3M as coagulant into PCR products to be purified, then adding sodium acetate and pre-cooled absolute ethanol solution added with polyethylene glycol 8000 respectively, so that the volume of polyethylene glycol 8000 in the final sample is 2.7% -20%, further promoting nucleic acid precipitation in the PCR products to be purified, and obtaining purified nucleic acid by centrifugal separation. The method solves the problem of the high concentration of the recovered product, improves the stability of the whole recovery process, reduces the variation coefficient of the concentration of the recovered product and the variation coefficient of the target strip area to be within 5 percent, and has the advantages of simple operation, high cost performance, high actual concentration and purity of the recovered product and good stability of the whole process.
Description
Technical Field
The application relates to the technical field of molecular biology, in particular to a method for recovering nucleic acid in PCR products by an ethanol-sodium acetate method.
Background
The recovery of the product is a key ring in a molecular cloning experiment, different recovery methods can influence the concentration and purity of the product, the key of success of a downstream molecular experiment is whether the actual concentration and purity of the recovered product reach the standard (A260/280 and A260/230 are about 1.80, A260/230 is used for evaluating whether a sample contains some pollutants such as carbohydrate, polypeptide, phenol, ethanol and the like, A260/280 is used for evaluating whether the purity of the sample is good, if protein pollution or RNA pollution and the like exist, the purity of the recovered product is low or the concentration of the product is high, the success rate of the connection and enzyme digestion experiment is reduced, the recovery amount is small, the workload is increased, and the efficiency is reduced. The common nucleic acid recovery methods mainly comprise a kit column recovery method and an ethanol-sodium acetate precipitation method. The recovery method of the kit column mainly achieves the recovery purpose through special adsorption buffer solution, elution buffer solution and DNA adsorption column, the operation of the kit is not complex, the recovery efficiency is moderate, but the single cost is high, taking the general DNA recovery kit DP214-02 of radix angelicae as an example, the single recovery cost is 6-8 RMB, the A260/230 value of the recovery product of the kit is generally poor, and the success rate of the connection and enzyme digestion experiment can be reduced. Meanwhile, the column type recovery has limitation on the total amount of recovered samples, recovery rate and recovery purity cannot be guaranteed for recovery of a large number of samples, and if the existing kit method is used for fractional recovery, high time cost and economic cost are required.
Ethanol-sodium acetate precipitation is based primarily on the fact that ethanol exposes negatively charged phosphate groups to which sodium ions can bind, reducing the rejection between polynucleotide chains at the site of precipitate formation. When the charges of the exposed phosphate groups are all balanced, nucleic acid changes from hydrophilicity to hydrophobicity to precipitate, the precipitation can be promoted to a certain extent at low temperature, the method is suitable for the recovery of linear carriers and a large number of PCR products, and although the method is suitable for the recovery of a large number of samples, the concentration of the PCR products recovered according to the prior method is poor in reality, namely the problem of high concentration deficiency exists, the recovery process has general stability, the follow-up molecular experiment is easily delayed due to the defects, and the working efficiency is reduced. The conventional ethanol-sodium acetate recovery PCR product has the defect of high concentration deficiency (mainly reflected in that the target strip area is not consistent with the actual concentration during gel running) and the problem of poor recovery process stability, and the defects can influence the recovery efficiency of the PCR product and the success rate of downstream molecular experiments.
Disclosure of Invention
In order to solve the technical problems, the application provides a method for recovering nucleic acid in a PCR product by an ethanol-sodium acetate method, which comprises the steps of adding guanidine hydrochloride with a final concentration of 1-3M into the PCR product to be purified as a coagulant, then respectively adding sodium acetate and a precooled absolute ethanol solution added with polyethylene glycol 8000, so that the volume of the polyethylene glycol 8000 in a final sample is 2.7-20%, further promoting nucleic acid precipitation in the PCR product to be purified, and then obtaining purified nucleic acid by centrifugal separation.
In one embodiment, guanidine hydrochloride is added as a setting accelerator at a final concentration of 1.5-2.5M.
In one embodiment, the purified nucleic acid is obtained by horizontal rotor centrifugation.
In one embodiment, 1-2 of M guanidine hydrochloride is added as a coagulant and the purified nucleic acid is obtained by horizontal rotor centrifugation.
In one embodiment, the method comprises the steps of:
step 1: adding guanidine hydrochloride with the final concentration of 1-3M into the PCR product to be purified as a coagulant and sodium acetate, and mixing the mixture upside down;
step 2: adding a precooled absolute ethyl alcohol solution added with polyethylene glycol 8000 to enable the volume of the polyethylene glycol 8000 in a final sample to be 2.7-20%, mixing the mixture upside down, and standing the mixture at a low temperature;
step 3: centrifuging by a centrifuge, removing supernatant liquid, and sucking all liquid drops on the pipe wall;
step 4: adding precooled 70% ethanol, gently mixing, centrifuging, removing supernatant, and sucking off residual liquid drop on the tube wall;
step 5: the purified nucleic acid obtained by centrifugation is placed in an oven for drying, and the residual liquid is volatilized to dryness.
In one embodiment, the centrifuge centrifugation in step 3 is a 4 ℃ horizontal centrifuge at 4000 rpm for 20 min.
In one embodiment, step 4 is repeated prior to step 5.
According to the application, the PCR product sedimentation promoter of 1-3M is added on the basis of a conventional ethanol-sodium acetate recovery process, the PCR product sedimentation promoter is matched in a horizontal centrifugal manner, the real concentration of the recovered PCR product is greatly improved through the synergistic effect between the PCR product sedimentation promoter and the PCR product sedimentation promoter, the A260/280 and A260/230 values of the purified product reach about 1.80, and finally the initial concentration of the PCR product is improved through the matched sedimentation of polyethylene glycol 8000 and ethanol, so that the loss during sample concentration is avoided, and the success of a downstream molecular experiment is facilitated and the working efficiency is improved. The method solves the problem of high concentration deficiency, improves the stability of the whole recovery process, reduces the variation coefficient of the sample concentration and the variation coefficient of the target strip area to be within 5 percent, and has simple operation, high cost performance, high actual concentration and good purity of the recovered product and good stability of the whole process.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the result of agarose electrophoresis comparing the method provided by the application with the conventional ethanol-sodium acetate precipitation method.
Detailed Description
In order that those skilled in the art will better understand the technical solutions of the present application, the present application will be further described with reference to examples. It will be apparent that the described embodiments are only some, but not all, embodiments of the application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, shall fall within the scope of the application. In the following examples, unless otherwise indicated, all methods conventional in the art are described.
Example one experiment of the effect of coagulants on different PCR products
Taking 12 tubes of 50 ul PCR products to be purified (agarose gel electrophoresis is verified to be a single strip and the same batch), wherein 3 tubes are not added with coagulant, and the other 9 tubes are respectively added with 3M guanidine hydrochloride, hydrochloric acid and sodium hydroxide, so that the final concentration of a single tube is 1M, each 3 tubes are parallel, then adding 3M sodium acetate with 1/10 total volume, and uniformly mixing the two tubes upside down; adding 2 times of precooled absolute ethyl alcohol, mixing uniformly, standing at-20 ℃ for 1 h, performing centrifugation according to a conventional centrifugation mode (angle rotor 4 ℃ C., 12000 rpm for 20 min), carefully removing supernatant after centrifugation, sucking residual liquid drops on the tube wall, adding 0.2 ml precooled 70% ethyl alcohol, gently mixing uniformly, centrifuging for 10 min, carefully removing supernatant, sucking residual liquid drops on the tube wall, and repeating the 70% ethyl alcohol rinsing once; the uncapped centrifuge tube is placed in a 50 ℃ oven for 15-20 min, residual liquid is volatilized to dryness, proper preheated enzyme-free water or TE buffer is added to dissolve DNA precipitate, the precipitate is quickly dissolved, and then the concentration is measured.
TABLE 1 influence of different product coagulants on the true concentration of PCR products
In table 1, the percentage of the area/concentration of the destination band: conversion was performed with the band area/concentration values of the conventional pellet as blank (conventional 100%); the higher the true concentration of the sample, the larger the area of the corresponding target strip, otherwise, the phenomenon of the concentration deficiency is considered, which is unfavorable for the smooth downstream experiment.
TABLE 2 influence of different product coagulants on the stability of recovery of PCR products
In the table above, CV (%) =standard deviation (SD)/average value is 100%. The coefficient of variation CV is used to reflect the degree of dispersion of the variable values and also the magnitude of the random error of the measurement results, with lower values indicating better process stability.
As shown by the result, when guanidine hydrochloride is added on the basis of conventional ethanol-sodium acetate recovery, the real concentration of the PCR product can be improved, the area and concentration of the strip are improved by 1.48 times compared with those of the conventional PCR product, and the problem that the concentration of the PCR product is virtually high is solved to a certain extent.
EXAMPLE two different molar concentration product accelerator experiments
Selecting guanidine hydrochloride in the first embodiment as a PCR product coagulant, optimizing a process based on the guanidine hydrochloride, taking 6 tubes of 50 ul PCR products to be purified (agarose gel electrophoresis is verified to be a single strip and the same batch), respectively adding 4M guanidine hydrochloride with different volumes, so that the final concentration of guanidine hydrochloride in a system is respectively 0.5M, 1M, 1.5M, 2M, 2.5M and 3M, then adding 3M sodium acetate with 1/10 total volume, and uniformly mixing the mixture upside down; adding 2 times of precooled absolute ethyl alcohol, mixing uniformly, standing at-20 ℃ for 1 h, performing centrifugation according to a conventional centrifugation mode (angle rotor 4 ℃ C., 12000 rpm for 20 min), carefully removing supernatant after centrifugation, sucking residual liquid drops on the tube wall, adding 0.2 ml precooled 70% ethyl alcohol, gently mixing uniformly, centrifuging for 10 min, carefully removing supernatant, sucking residual liquid drops on the tube wall, and repeating the 70% ethyl alcohol rinsing once; the uncapped centrifuge tube is placed in a 50 ℃ oven for 15-20 min, residual liquid is volatilized to dryness, proper preheated enzyme-free water or TE buffer is added to dissolve DNA precipitate, the precipitate is quickly dissolved, and then the concentration is measured.
TABLE 3 influence of different molar concentrations of product coagulants on recovery and purity of PCR products
In table 3 above, the percentage of the area/concentration of the destination band: conversion was performed with the band area/concentration values of the conventional pellet as blank (conventional 100%); the higher the true concentration of the sample, the larger the area of the corresponding target strip, otherwise, the phenomenon of the concentration deficiency is considered, which is unfavorable for the smooth downstream experiment.
As shown by the result, when the final concentration of guanidine hydrochloride is 1-3M, the preferable concentration range is 1.5-2.5M, the recovery yield and purity of the PCR product are improved to a certain extent, the area of the strip and the concentration of the PCR product can be improved by 3.21 times as compared with the conventional method, the condition that the concentration of the conventional sodium acetate recovery process is high in a deficiency way is greatly reduced, and the concentration authenticity is enhanced, so that the method is favorable for downstream experiments such as enzyme digestion and connection experiments.
Example three different centrifugation mode comparison experiments
Taking 6 tubes of 50-ul PCR products to be purified (agarose gel electrophoresis verifies that the PCR products are single strips and the same batch), adding 1/10 total volume of 3-M sodium acetate into each tube, and mixing the mixture upside down; adding 2 times of pre-cooled absolute ethyl alcohol, uniformly mixing, placing at-20 ℃ for 1 h, performing centrifugation according to different centrifugation modes (the centrifugation of a front 3-tube horizontal rotor is performed for 20 min at 4 ℃ and 4000 rpm, the centrifugation of a rear 3-tube angular rotor is performed for 20 min at 4 ℃ and 12000 rpm), carefully removing the supernatant after the centrifugation is finished, sucking off residual liquid drops on the tube wall, adding 0.2 ml of pre-cooled 70% ethyl alcohol, gently mixing, centrifuging for 10 min, carefully removing the supernatant, sucking off residual liquid drops on the tube wall, and repeating the 70% ethyl alcohol rinsing once; the uncapped centrifuge tube is placed in a 50 ℃ oven for 15-20 min, residual liquid is volatilized to dryness, proper preheated enzyme-free water or TE buffer is added to dissolve DNA precipitate, the precipitate is quickly dissolved, and then the concentration is measured.
TABLE 4 influence of different centrifugation modes on the recovery purity and reproducibility of PCR products
In the table, the higher the true concentration of the sample, the larger the area of the corresponding target strip, otherwise, the phenomenon of the concentration deficiency is considered, the smooth downstream experiment is not facilitated, the variation coefficient CV is used for reflecting the discrete degree of the variable value, the size of the random error of the measurement result is also reflected, and the lower the value is, the better the process stability is indicated.
According to the result, when the conventional ethanol-sodium acetate recovery centrifugal mode is replaced, namely the centrifugal mode is replaced by the horizontal rotor centrifugal mode from the angular rotor centrifugal mode, the ratio of the target strip area to the concentration of the PCR product can be improved by 1.74 times, the ratio is improved to 73.57 from 42.28, the phenomenon of the virtual high concentration can be effectively reduced compared with the conventional method, meanwhile, the repeatability of the PCR product recovery is improved to a certain extent, the variation coefficient of the concentration of the purified sample and the variation coefficient of the target strip area are reduced to be within 5%, the stability of the horizontal rotor recovery process is better, and the horizontal rotor centrifugal mode is selected as the main centrifugal mode of the recovery process.
Example four experiments with guanidine hydrochloride and horizontal rotor at different molar concentrations
Selecting the horizontal rotor centrifugation in the third embodiment as a main centrifugation mode, taking 8 tubes of 50 ul PCR products to be purified (agarose gel electrophoresis is verified as a single strip and the same batch), setting the PCR products into 2 groups, respectively adding 4 groups of 4 tubes of samples into the 2 groups of samples, respectively adding 4M guanidine hydrochloride to ensure that the final concentration of single-tube guanidine hydrochloride in the groups is 1M, 1.5M, 2M and 2.5M respectively, then adding 3M sodium acetate with 1/10 total volume into each tube, and uniformly mixing the mixture upside down; adding 2 times of pre-cooled absolute ethyl alcohol, mixing uniformly, standing at-20 ℃ for 1 h, performing centrifugation according to different centrifugation modes (centrifugation of a group 1 angle rotor is performed for 20 min at 4 ℃ and 12000 rpm, centrifugation of a group 2 horizontal rotor is performed for 20 min at 4 ℃ and 4000 rpm), carefully removing supernatant after centrifugation is finished, sucking residual liquid drops on the tube wall, adding 0.2 ml of pre-cooled 70% ethyl alcohol, gently mixing, centrifuging for 10 min, carefully removing supernatant, sucking residual liquid drops on the tube wall, and repeating the 70% ethyl alcohol rinsing once; the uncapped centrifuge tube is placed in a 50 ℃ oven for 15-20 min, residual liquid is volatilized to dryness, proper preheated enzyme-free water or TE buffer is added to dissolve DNA precipitate, the precipitate is quickly dissolved, and then the concentration is measured.
TABLE 5 influence of different centrifugation modes in combination with guanidine hydrochloride on recovery and purity of PCR products
In the above table, the percentage of the area/concentration of the band of interest: conversion was performed with the band area/concentration values of the conventional pellet as blank (conventional 100%); the higher the true concentration of the sample, the larger the area of the corresponding target strip, otherwise, the phenomenon of the concentration deficiency is considered, which is unfavorable for the smooth downstream experiment.
As shown by the result, when the accelerator guanidine hydrochloride of the PCR product is centrifugally matched with the horizontal rotor, the ratio of the target strip area to the concentration of the PCR product can be improved by 4.29 times to the maximum, the real concentration of the PCR product is greatly improved, the phenomenon of deficiency and high concentration can be effectively reduced, and compared with the single guanidine hydrochloride addition or the single centrifugal replacement mode, the accelerator guanidine hydrochloride of the PCR product is better in matching effect, so that the waste liquid can be conveniently and controllably removed after the PCR product is gathered from the pipe wall to the pipe bottom, and the product loss during the waste liquid removal can be effectively reduced. Meanwhile, the change of the precipitation part may have a certain positive effect on the subsequent ethanol rinsing and residual ethanol volatilization, and the synergistic effect of the PCR product coagulant guanidine hydrochloride is added, so that the real concentration of the PCR recovery is improved, and the subsequent PCR product recovery process is preferably matched with horizontal rotor centrifugation by using guanidine hydrochloride.
Fifth example experiment of the precipitation effect of polyethylene glycol 8000 with different volume concentrations
In the fourth selected example, guanidine hydrochloride with the final concentration of 1.5M is added as the basis, and is matched in a horizontal centrifugal way, and as the basis of the control experiment of the round, 7 tubes of 50 ul PCR products to be purified (agarose gel electrophoresis is verified to be a single strip and the same batch) are taken, 4M guanidine hydrochloride is added, so that the final concentration of guanidine hydrochloride is 1.5M, then 3M sodium acetate with the total volume of 1/10 is added into each tube, and the mixture is uniformly mixed upside down; then, absolute ethanol added with polyethylene glycol 8000 is added into 7-tube samples respectively, so that the volumes (v/v) of the polyethylene glycol 8000 in the final samples are respectively 0%, 2.7%, 6.7%, 13.3%, 20.0%, 26.7% and 33.3%, and the 7-tube samples are placed at-20 ℃ for 1 h, and are centrifuged by using a horizontal rotor: centrifuging at 4deg.C for 20 min at 4000 rpm, carefully removing supernatant after centrifuging, sucking off residual liquid drop on the tube wall, adding 70% ethanol precooled by 0.2 ml, gently mixing, centrifuging for 10 min, carefully removing supernatant, sucking off residual liquid drop on the tube wall, and repeating 70% ethanol rinsing once; the uncapped centrifuge tube is placed in a 50 ℃ oven for 15-20 min, residual liquid is volatilized to dryness, proper preheated enzyme-free water or TE buffer is added to dissolve DNA precipitate, the precipitate is quickly dissolved, and then the concentration is measured.
TABLE 6 influence of different volumes of polyethylene glycol 8000 on the recovery concentration of PCR products by the experiments of the precipitation effect
In the above table, the percentage of the area/concentration of the band of interest: conversion was performed with the band area/concentration values of the conventional pellet as blank (conventional 100%); the higher the true concentration of the sample, the larger the area of the corresponding target strip, otherwise, the phenomenon of the concentration deficiency is considered, which is unfavorable for the smooth downstream experiment.
According to the results, when the volume of polyethylene glycol 8000 in the absolute ethanol solution is 2.7-20% (v/v), the initial concentration and the real concentration of the recovered PCR product are improved to a certain extent, the area and the concentration of the strip can be improved by 4.16 times as compared with the conventional recovery process, meanwhile, the condition of the concentration deficiency of the conventional sodium acetate recovery process can be reduced to a certain extent by adding polyethylene glycol 8000, and the method provided by the application is higher in the recovery initial concentration, so that the sample loss in the concentration of the subsequent sequencing experiment is avoided. The subsequent PCR product recovery process is therefore preferably performed using an absolute ethanol solution with the addition of polyethylene glycol 8000 such that the polyethylene glycol volume in the final sample is 2.7-20%.
EXAMPLE six conventional ethanol-sodium acetate method and method of the application for recovery of PCR products comparative experiments
Selecting an ethanol-sodium acetate precipitation process which is optimized finally and comprises the steps of adding guanidine hydrochloride with a final concentration of 1.5M into a recovery process, matching in a horizontal centrifugal manner, adding polyethylene glycol 8000 into the solution, taking 8 tubes of 50 ul PCR products to be purified (agarose gel electrophoresis is verified to be single strip), dividing the 4 tubes into two batches, adding guanidine hydrochloride with a final concentration of 1.5M into 2 tubes in batch 1, adding guanidine hydrochloride into 2 tubes, adding 3M sodium acetate with a total volume of 1/10, and uniformly mixing the two batches upside down; adding pre-cooled absolute ethyl alcohol with the total volume being 2 times that of a sample without guanidine hydrochloride, uniformly mixing, adding absolute ethyl alcohol solution with the polyethylene glycol 8000 with the total volume being 2 times that of the sample without guanidine hydrochloride, uniformly mixing, finally adding 13.3% of polyethylene glycol 8000 in the sample, placing 1 h at the temperature of-20 ℃ for 4-pipe samples, performing centrifugation according to different centrifugation modes (centrifugation of a horizontal rotor of the sample without guanidine hydrochloride: centrifugation at the temperature of 4 ℃ for 20 min,4000 rpm, centrifugation of an angular rotor of the sample without guanidine hydrochloride: centrifugation at the temperature of 4 ℃ for 20 min,12000 rpm), carefully removing the supernatant after centrifugation is finished, sucking off residual liquid drops on pipe walls, adding 70% ethanol with the pre-cooled temperature of 0.2 ml, gently mixing, carefully removing the supernatant after centrifugation for 10 min, sucking off residual liquid drops on the pipe walls, and repeating the 70% ethanol rinsing once; the uncapped centrifuge tube is placed in a 50 ℃ oven for 15-20 min, residual liquid is volatilized to dryness, proper preheated enzyme-free water or TE buffer is added to dissolve DNA precipitate, rapid dissolution of the precipitate is seen, then the concentration is measured and gel is run, the batch 1 is obtained, and the rest 4 tubes of PCR products to be purified are recycled again as batch 2 in the same manner. FIG. 1 shows the comparison of agarose electrophoresis results of the method provided by the application with those of a conventional ethanol-sodium acetate precipitation method, wherein samples recovered by two methods of batch 1 are selected, 1 ul samples are respectively taken and loaded, and 1 ul is added into a 5000 bp Marker.
TABLE 7 comparison of conventional ethanol-sodium acetate method with the recovery of PCR products by the method of the present application
In the table, the higher the true concentration of the sample is, the larger the area of the corresponding target strip is, otherwise, the phenomenon of the concentration deficiency is considered, and the downstream experiment is not easy to be carried out smoothly; the coefficient of variation CV is used to reflect the degree of dispersion of the variable values and also the magnitude of the random error of the measurement results, with lower values indicating better process stability.
Compared with the conventional sodium acetate precipitation method, the optimized sodium acetate recovery method can effectively improve the real concentration of the PCR product recovery, the ratio of the strip area to the concentration can be improved to 175.50 from 47.16 by about 3.72 times, the recovery rate of the PCR product is improved, meanwhile, the purity A260/280 and A260/230 of the product recovered by the method can reach about 1.80, the problem that the ratio of the product A260/230 recovered by the conventional PCR product recovery kit is low is solved, the concentration authenticity is enhanced, the development of downstream experiments is facilitated by better A260/280 and A260/230 values, the variation coefficient of the concentration of the whole samples of two batches and the variation coefficient of the target strip area are improved to a certain extent to within 5% compared with the conventional method, and the feasibility and the stability of the optimized recovery method are reflected.
It is to be understood that this application is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present application which will be limited only by the appended claims.
Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the application described herein. Such equivalents are also encompassed by the appended claims.
Claims (7)
1. The method for recovering nucleic acid in PCR product by using ethyl alcohol-sodium acetate method is characterized by that guanidine hydrochloride whose final concentration is 1-3M is added into the PCR product to be purified as coagulant, then sodium acetate and precooled absolute ethyl alcohol solution to which polyethylene glycol 8000 is added are respectively added so as to make the volume of polyethylene glycol 8000 in the final sample be 2.7% -20%, further promote nucleic acid precipitation in the PCR product to be purified, and then the purified nucleic acid can be obtained by means of centrifugal separation.
2. The method according to claim 1, wherein guanidine hydrochloride is added as a setting accelerator in a final concentration of 1.5-2.5. 2.5M.
3. The method of claim 1, wherein the purified nucleic acid is obtained by horizontal rotor centrifugation.
4. The method according to claim 3, wherein the purified nucleic acid is obtained by centrifugation on a horizontal rotor by adding 1-2M guanidine hydrochloride as a coagulant.
5. The method according to any one of claims 1-4, characterized in that the method comprises the steps of:
step 1: adding guanidine hydrochloride with the final concentration of 1-3M into the PCR product to be purified as a coagulant and sodium acetate, and mixing the mixture upside down;
step 2: adding a precooled absolute ethyl alcohol solution added with polyethylene glycol 8000 to ensure that the volume concentration of the polyethylene glycol 8000 in a final sample is 2.7-20%, mixing the mixture upside down, and standing the mixture at a low temperature;
step 3: centrifuging by a centrifuge, removing supernatant liquid, and sucking all liquid drops on the pipe wall;
step 4: adding precooled 70% ethanol, gently mixing, centrifuging, removing supernatant, and sucking off residual liquid drop on the tube wall;
step 5: the purified nucleic acid obtained by centrifugation is placed in an oven for drying, and the residual liquid is volatilized to dryness.
6. The method according to claim 5, wherein the centrifuge centrifugation in step 3 is a horizontal centrifuge at 4℃at 4000 rpm for 20 min.
7. The method of claim 5, wherein step 4 is repeated prior to step 5.
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